Rat Liver 10-formyltetrahydrofolate Dehydrogenase, Carbamoyl Phosphate Synthetase 1 and Betaine Homocysteine S-methytransferase were Co-purified on Kunitz-type Soybean Trypsin Inhibitor-coupled Sepharose CL-4B

  • Published : 2007.07.31


An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.


  1. Bailey, L. B. and Gregory, J. F., 3rd (1999) Folate metabolism and requirements. J. Nutr. 129, 779-782.
  2. Cho, M. Y., Lee, H. S., Lee, K. M., Homma, K., Natori, S. and Lee, B. L. (1999) Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae. Eur. J. Biochem. 262, 737-744.
  3. Christeller, J. T. (2005) Evolutionary mechanisms acting on proteinase inhibitor variability. FEBS J. 272, 5710-5722.
  4. Chumanevich, A. A., Krupenko, S. A. and Davies, C. (2004) The crystal structure of the hydrolase domain of 10-formyltetrahydrofolate dehydrogenase: mechanism of hydrolysis and its interplay with the dehydrogenase domain. J. Biol. Chem. 279, 14355-14364.
  5. Cook, R. J., Lloyd, R. S. and Wagner, C. (1991) Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase. J. Biol. Chem. 266, 4965-4973.
  6. Guo, L., Enzan, H., Hayashi, Y., Miyazaki, E., Jin, Y., Toi, M., Kuroda, N. and Hiroi, M. (2006) Increased iron deposition in rat liver fibrosis induced by a high-dose injection of dimethylnitrosamine. Exp. Mol. Pathol. 81, 255-261.
  7. Huo, R., Zhu, H., Lu, L., Ying, L., Xu, M., Xu, Z., Li, J., Zhou, Z. and Sha, J. (2005) Molecular cloning, identification and characteristics of a novel isoform of carbamyl phosphate synthetase I in human testis. J. Biochem. Mol. Biol. 38, 28-33.
  8. Inglese, J., Smith, J. M. and Benkovic, S. J. (1990) Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli. Biochemistry 29, 6678-6687.
  9. Iwanaga, S., and Lee, B. L. (2005) Recent advances in the innate immunity of invertebrate animals. J. Biochem. Mol. Biol. 38, 128-150.
  10. Jacobs, R. L., Stead, L. M., Devlin, C., Tabas, I., Brosnan, M. E., Brosnan, J. T. and Vance, D. E. (2005) Physiological regulation of phospholipid methylation alters plasma homocysteine in mice. J. Biol. Chem. 280, 28299-28305.
  11. Ju, J. S., Cho, M. H., Brade, L., Kim, J. H., Park, J. W., Ha, N. C., Soderhall, I., Soderhall, K., Brade, H. and Lee, B. L. (2006) A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide. J. Immunol. 177, 1838-1845.
  12. Kothe, M., Purcarea, C., Guy, H. I., Evans, D. R. and Powers-Lee, S. G. (2005) Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase. Protein Sci. 14, 37-44.
  13. Krupenko, S. A., Wagner, C. and Cook, R. J. (1997) Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. Resolution of the amino-terminal domain as 10-formyltetrahydrofolate hydrolase. J. Biol. Chem. 272, 10273-10278.
  14. Lee, M. H., Osaki, T., Lee, J. Y., Baek, M. J., Zhang, R., Park, J. W., Kawabata, S., Soderhall, K. and Lee, B. L. (2004) Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect. J. Biol. Chem. 279, 3218-3227.
  15. Murthy, H. M., Clum, S. and Padmanabhan, R. (1999) Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J. Biol. Chem. 274, 5573-5580.
  16. Odani, S., Odani, S., Ono, T. and Ikenaka, T. (1979) Proteinase inhibitors from a mimosoideae legume, Albizzia julibrissin. Homologues of soybean trypsin inhibitor (Kunitz). J. Biochem (Tokyo) 86, 1795-1805.
  17. Pajares, M. A. and Perez-Sala, D. (2006) Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism? Cell Mol. Life Sci. 63, 2792-2803.
  18. Perona, J. J. and Craik, C. S. (1995) Structural basis of substrate specificity in the serine proteases. Protein Sci. 4, 337-360.
  19. Prezelj, A., Anderluh, P. S., Peternel, L. and Urleb, U. (2007) Recent advances in serine protease inhibitors as anticoagulant agents. Curr. Pharm. Des. 13, 287-312.
  20. Rawlings, N. D., Tolle, D. P. and Barrett, A. J. (2004) Evolutionary families of peptidase inhibitors. Biochem. J. 378, 705-716.
  21. Rubio, V. and Cervera, J. (1995) The carbamoyl-phosphate synthase family and carbamate kinase: structure-function studies. Biochem. Soc. Trans. 23, 879-883.

Cited by

  1. Proteomics and gene expression analyses of squalene-supplemented mice identify microsomal thioredoxin domain-containing protein 5 changes associated with hepatic steatosis vol.77, 2012,